The human astroviruses are emerging as an important cause of gastroenteritis in humans, particularly in young children, the elderly, and immunocompromised adults. Studies to define the determinants of astrovirus transmission and virulence have been hindered by a limited understanding of the mechanism and genetics of astrovirus replication. Specifically, it has not been possible to confirm the function of predicted astrovirus gene products during virus replication in cells. The overall goal of this pilot project is to demonstrate the feasibility of using a full-length cDNA clone of the human astrovirus genome RNA to investigate the genetics of astrovirus replication. The proposal will focus on the serine proteinase domain of human astrovirus serotype 2 (Hast-2) as a model for determining the function of astrovirus proteins. Experiments in Specific AIM 1 will define the function of the virus- encoded serine proteinase in processing of the astrovirus polymerase gene polyprotein. Astrovirus RNA obtained from infectious virions, from the in vitro transcribed full-length cDNA of the astrovirus genome, and from in vitro transcription of subclones of the polymerase gene 1, will be translated in vitro. The location and activity of the serine proteinase (Spro) in protein processing will be defined. Experiments in Specific AIM 2 will determine the effect of mutations in the viral proteinase on protein processing in vitro and on virus replication in cells. Mutations will be introduced at a putative catalytic residue of Spro within the full-length cDNA clone of the Hast-2 genome, and the effect on protein processing will be determined during in vitro translation of RNA transcribed from the cDNA. The in vitro transcribed mutant Hast-2 RNA will be used to transfect cells to define the effect on replication. Together these studies will illustrate the usefulness of the full-length Hast-2 cDNA as a tool to study all aspects of astrovirus biology, and will provide important insights into the life cycle of this emerging human pathogen.